Ophthalmic compositions for treating ocular hypertension

ABSTRACT

This invention relates to the use of potent potassium channel blockers or a formulation thereof in the treatment of glaucoma and other conditions which leads to elevated intraoccular pressure in the eye of a patient. This invention also relates to the use of such compounds to provide a neuroprotective effect to the eye of mammalian species, particularly humans.

This application is a 371 of PCT/US02/03049, filed Jan. 25, 2002 Whichclaims benefit from U.S. Provisional Application No. 60/264,954, filedJan. 30, 2001.

BACKGROUND OF THE INVENTION

Glaucoma is a degenerative disease of the eye wherein the intraocularpressure is too high to permit normal eye function. As a result, damagemay occur to the optic nerve head and result in irreversible loss ofvisual function. If untreated, glaucoma may eventually lead toblindness. Ocular hypertension, i.e., the condition of elevatedintraocular pressure without optic nerve head damage or characteristicglaucomatous visual field defects, is now believed by the majority ofophthalmologists to represent merely the earliest phase in the onset ofglaucoma.

Many of the drugs formerly used to treat glaucoma proved unsatisfactory.The early methods of treating glaucoma employed pilocarpine and producedundesirable local effects that made this drug, though valuable,unsatisfactory as a first line drug. More recently, clinicians havenoted that many β-adrenergic antagonists are effective in reducingintraocular pressure. While many of these agents are effective for thispurpose, there exist some patients with whom this treatment is noteffective or not sufficiently effective. Many of these agents also haveother characteristics, e.g., membrane stabilizing activity, that becomemore apparent with increased doses and render them unacceptable forchronic ocular use and can also cause cardiovascular effects.

Although pilocarpine and β-adrenergic antagonists reduce intraocularpressure, none of these drugs manifests its action by inhibiting theenzyme carbonic anhydrase, and thus they do not take advantage ofreducing the contribution to aqueous humor formation made by thecarbonic anhydrase pathway.

Agents referred to as carbonic anhydrase inhibitors decrease theformation of aqueous humor by inhibiting the enzyme carbonic anhydrase.While such carbonic anhydrase inhibitors are now used to treatintraocular pressure by systemic and topical routes, current therapiesusing these agents, particularly those using systemic routes are stillnot without undesirable effects. Because carbonic anhydrase inhibitorshave a profound effect in altering basic physiological processes, theavoidance of a systemic route of administation serves to diminish, ifnot entirely eliminate, those side effects caused by inhibition ofcarbonic anhydrase such as metabolic acidosis, vomiting, numbness,tingling, general malaise and the like. Topically effective carbonicanhydrase inhibitors are disclosed in U.S. Pat. Nos. 4,386,098;4,416,890; 4,426,388; 4,668,697; 4,863,922; 4,797,413; 5,378,703,5,240,923 and 5,153,192.

Prostaglandins and prostaglandin derivatives are also known to lowerintraocular pressure. U.S. Pat. No. 4,883,819 to Bito descibes the useand synthesis of PGAs, PGBs and PGCs in reducing intraocular pressure.U.S. Pat. No. 4,824,857 to Goh et al. describes the use and synthesis ofPGD2 and derivatives thereof in lowering intraocular pressure includingderivatives wherein C-10 is replaced with nitrogen. U.S. Pat. No.5,001,153 to Ueno et al. describes the use and synthesis of13,14-dihydro-15-keto prostaglandins and prostaglandin derivatives tolower intraocular pressure. U.S. Pat. No. 4,599,353 describes the use ofeicosanoids and eicosanoid derivatives including prostaglandins andprostaglandin inhibitors in lowering intraocular pressure.

Prostaglandin and prostaglandin derivatives lower intraocular pressureby increasing uveoscleral outflow. This is true for both the F type andA type of Pgs and hence presumably also for the B, C, D, E and J typesof prostaglandins and derivatives thereof. A problem with usingprostaglandin derivatives to lower intraocular pressure is that thesecompounds often induce an initial increase in intraocular pressure, canchange the color of eye pigmentation and cause proliferation of sometissues surrounding the eye.

As can be seen, there are several current therapies for treatingglaucoma and elevated intraocular pressure, but the efficacy and theside effect profiles of these agents are not ideal. Recently potassiumchannel blockers were found to reduce intraocular pressure in the eyeand therefore provide yet one more approach to the treatment of ocularhypertension and the degenerative ocular conditions related thereto.Blockage of potassium channels can diminish fluid secretion, and undersome circumstances, increase smooth muscle contraction and would beexpected to lower IOP and have neuroprotective effects in the eye. (seeU.S. Pat. Nos. 5,573,758 and 5,925,342; Moore, et al., Invest.Ophthalmol. Vis. Sci 38, 1997; WO 89/10757, WO94/28900, and WO96/33719).

SUMMARY OF THE INVENTION

This invention relates to potent potassium channel blockers, their useor a formulation thereof in the treatment of glaucoma and otherconditions which are related to elevated intraocular pressure in the eyeof a patient. This invention also relates to the use of such compoundsto provide a neuroprotective effect to the eye of mammalian species,particularly humans. More particularly this invention relates to thetreatment of glaucoma and/or ocular hypertension (elevated intraocularpressure) using ethanecarbothioic S acid ester compounds having thestructural formula I:

or a pharmaceutically acceptable salt, enantiomer, diastereomer ormixture thereof:

-   -   wherein,    -   R and R² independently represent C₁₋₆ alkyl, (CH₂)_(n)aryl,        (CH₂)_(n)heteroaryl, (CH₂)_(n)heterocycloalkyl, said alkyl, aryl        or heteroaryl optionally substituted with 1-3 groups of R³;    -   Y represents —(CH₂)_(n)SCOR⁴;    -   X represents CH₂, or O (in which m does not exist);    -   R³ represents hydrogen, C₁₋₆ alkoxy, C₁₋₆ alkyl, CF₃, nitro,        amino, cyano, C₁₋₆ alkylamino, or halogen and    -   R⁴ represents C₁₋₆ alkoxy, or C₁₋₆ alkyl;    -   R⁷ represents H, halo, OH, NO₂, NH₂, CN, alkoxy, —COO—,        alkoxycarbonyl, haloalkyl, alkoxycarbonylalkyl, or        alkylsulphonyl;    -   m represents 1-3; and/or    -   n represents 0-3.

This and other aspects of the invention will be realized upon inspectionof the invention as a whole.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to a method for decreasing elevatedintraocular pressure or treating glaucoma by administration, preferablytopical or intra-camaral administration, of a composition containing apotassium channel blocker of Formula I and a pharmaceutically acceptablecarrier.

One embodiment of this invention is realized when R is C₁₋₆ alkyl, or(CH₂)_(n) aryl, and all other variables are as originally described.

Another embodiment of this invention is realized when R₂ is C₁₋₆ alkyl,or (CH₂)_(n) aryl, and all other variables are as originally described.

Yet another embodiment of this invention is realized when X is CH₂ andall other variables are as originally described.

Still another embodiment of this invention is realized when Y is—(CH₂)_(n)SCOR⁴ wherein n=0, and all other variables are as originallydescribed.

Another embodiment of this invention is realized when Y is—(CH₂)_(n)SCOR⁴, wherein n=1-3, and all other variables are asoriginally described.

A preferred embodiment of this invention is realized when R is (CH₂)_(n)aryl, R² is C₁₋₆ alkyl, Y is (CH₂)_(n)SCOR⁴, X is CH₂ and m=1.

Another preferred embodiment of this invention is realized when R is(CH₂)_(n)aryl, R² is C₁₋₆ alkyl, Y is (CH₂)_(n)SCOR⁴, X is CH₂ and m=2.

Still another preferred embodiment of this invention is realized when Ris C₁₋₆ alkyl, R² is (CH₂)_(n)aryl, Y is (CH₂)_(n)SCOR⁴, X is CH₂ andm=2.

Yet another preferred embodiment of this invention is realized when R is(CH₂)_(n)aryl, R² is (CH₂)_(n)aryl, Y is (CH₂)_(n)SCOR⁴, X is CH₂ andm=2.

Yet another preferred embodiment of this invention is realized when R⁷is H and all other variables are as originally described.

Preferred compounds of this invention are:

The invention is described herein in detail using the terms definedbelow unless otherwise specified.

The term “alkyl” refers to a monovalent alkane (hydrocarbon) derivedradical containing from 1 to 10 carbon atoms unless otherwise defined.It may be straight, branched or cyclic. Preferred alkyl groups includemethyl, ethyl, propyl, isopropyl, butyl, t-butyl, cyclopentyl andcyclohexyl. When the alkyl group is said to be substituted with an alkylgroup, this is used interchangeably with “branched alkyl group”.

Cycloalkyl is a specie of alkyl containing from 3 to 15 carbon atoms,without alternating or resonating double bonds between carbon atoms. Itmay contain from 1 to 4 rings which are fused.

Alkoxy refers to C₁-C₆ alkyl-O—, with the alkyl group optionallysubstituted as described herein.

Halogen (halo) refers to chlorine, fluorine, iodine or bromine.

Aryl refers to aromatic rings e.g., phenyl, substituted phenyl and thelike, as well as rings which are fused, e.g., naphthyl, phenanthrenyland the like. An aryl group thus contains at least one ring having atleast 6 atoms, with up to five such rings being present, containing upto 22 atoms therein, with alternating (resonating) double bonds betweenadjacent carbon atoms or suitable heteroatoms. The preferred aryl groupsare phenyl, naphthyl and phenanthrenyl. Aryl groups may likewise besubstituted as defined. Preferred substituted aryls include phenyl andnaphthyl.

The term “heterocycloalkyl” refers to a cycloalkyl group (nonaromatic)in which one of the carbon atoms in the ring is replaced by a heteroatomselected from O, S or N, and in which up to three additional carbonatoms may be replaced by hetero atoms.

The term “heteroatom” means O, S or N, selected on an independent basis.

The term “heteroaryl” refers to a monocyclic aromatic hydrocarbon grouphaving 5 or 6 ring atoms, or a bicyclic aromatic group having 8 to 10atoms, containing at least one heteroatom, O, S or N, in which a carbonor nitrogen atom is the point of attachment, and in which one or twoadditional carbon atoms is optionally replaced by a heteroatom selectedfrom O or S, and in which from 1 to 3 additional carbon atoms areoptionally replaced by nitrogen heteroatoms, said heteroaryl group beingoptionally substituted as described herein. Examples of this type arepyrrole, pyridine, oxazole, thiazole and oxazine. Additional nitrogenatoms may be present together with the first nitrogen and oxygen orsulfur, giving, e.g., thiadiazole.

This invention is also concerned with a method of treating ocularhypertension or glaucoma by administering to a patient in need thereofone of the compounds of formula I in combination with a β-adrenergicblocking agent such as timolol, a parasympathomimetic agent such aspilocarpine, carbonic anhydrase inhibitor such as dorzolamide,acetazolamide, metazolamide or brinzolamide, a prostaglandin such aslatanoprost, rescula, S1033 or a hypotensive lipid derived from PGF2αprostaglandins such as prostamide (AGN 192024). An example of ahypotensive lipid (the carboxylic acid group on the α-chain link of thebasic prostaglandin structure is replaced with electrochemically neutralsubstituents) is that in which the carboxylic acid group is replacedwith a C₁₋₆ alkoxy group such as OCH₃ (PGF_(2a) 1-OCH₃), or a hydroxygroup (PGF_(2a) 1-OH).

Preferred potassium channel blockers are calcium activated potassiumchannel blockers. More preferred potassium channel blockers are highconductance, calcium activated potassium (Maxi-K) channel blockers.Maxi-K channels are a family of ion channels that are prevalent inneuronal, endocrine smooth muscle and epithelial tissues and which aregated by membrane potential and intracellular Ca²⁺.

Intraocular pressure (IOP) is controlled by aqueous humor dynamics.Aqueous humor is produced at the level of the non-pigmented ciliaryepithelium and is cleared primarily via outflow through the trabecularmeshwork. Aqueous humor inflow is controlled by ion transport processes.It is thought that maxi-K channels in non-pigmented ciliary epithelialcells indirectly control chloride secretion by two mechanisms; thesechannels maintain a hyperpolarized membrane potential (interiornegative) which provides a driving force for chloride efflux from thecell, and they also provide a counter ion (K⁺) for chloride ionmovement. Water moves passively with KCl allowing production of aqueoushumor. Inhibition of maxi-K channels in this tissue would diminishinflow. Maxi-K channels have also been shown to control thecontractility of certain smooth muscle tissues, and, in some cases,channel blockers can contract quiescent muscle, or increase the myogenicactivity of spontaneously active tissue. Contraction of ciliary musclewould open the trabecular meshwork and stimulate aqueous humor outflow,as occurs with pilocarpine. Therefore maxi-K channels could profoundlyinfluence aqueous humor dynamics in several ways; blocking this channelwould decrease IOP by affecting inflow or outflow processes or by acombination of affecting both inflow/outflow processes.

The present invention is based upon the finding that maxi-K channels, ifblocked, inhibit aqueous humor production by inhibiting net solute andH₂O efflux and therefore lower IOP. This finding suggests that maxi-Kchannel blockers are useful for treating other ophthamologicaldysfunctions such as macular edema and macular degeneration. It is knownthat lowering IOP promotes blood flow to the retina and optic nerve.Accordingly, the compounds of this invention are useful for treatingmacular edema and/or macular degeneration.

Macular edema is swelling within the retina within the criticallyimportant central visual zone at the posterior pole of the eye. Anaccumulation of fluid within the retina tends to detach the neuralelements from one another and from their local blood supply, creating adormancy of visual function in the area.

Glaucoma is characterized by progressive atrophy of the optic nerve andis frequently associated with elevated intraocular pressure (IOP). It ispossible to treat glaucoma, however, without necessarily affecting IOPby using drugs that impart a neuroprotective effect. See Arch.Ophthalmol. Vol. 112, January 1994, pp. 37-44; Investigative Ophthamol.& Visual Science, 32, 5, April 1991, pp. 1593-99. It is believed thatmaxi-K channel blockers which lower IOP are useful for providing aneuroprotective effect. They are also believed to be effective forincreasing retinal and optic nerve head blood velocity and increasingretinal and optic nerve oxygen by lowering IOP, which when coupledtogether benefits optic nerve health. As a result, this inventionfurther relates to a method for increasing retinal and optic nerve headblood velocity, increasing retinal and optic nerve oxygen tension aswell as providing a neuroprotective effect or a combination thereof.

As indicated above, potassium channel antagonists are useful for anumber of physiological disorders in mammals, including humans. Ionchannels, including potassium channels, are found in all mammalian cellsand are involved in the modulation of various physiological processesand normal cellular homeostasis. Potassium ions generally control theresting membrane potential, and the efflux of potassium ions causesrepolarization of the plasma membrane after cell depolarization.Potassium channel antagonists prevent repolarization and enable the cellto stay in the depolarized, excited state.

There are a number of different potassium channel subtypes.Physiologically, one of the most important potassium channel subtypes isthe Maxi-K channel which is present in neuronal and endocrine tissue,smooth muscle and epithelial tissue. Intracellular calcium concentration(Ca²⁺ _(i)) and membrane potential gate these channels. For example,Maxi-K channels are opened to enable efflux of potassium ions by anincrease in the intracellular Ca²⁺ concentration or by membranedepolarization (change in potential). Elevation of intracellular calciumconcentration is required for neurotransmitter release. Modulation ofMaxi-K channel activity therefore affects transmitter release from thenerve terminal by controlling membrane potential, which in turn affectsthe influx of extracellular Ca²⁺ through voltage-gated calcium channels.The compounds of the present invention are therefore useful in thetreatment of neurological disorders in which neurotransmitter release isimpaired.

A number of marketed drugs function as potassium channel antagonists.The most important of these include the compounds Glyburide, Glipizideand Tolbutamide. These potassium channel antagonists are useful asantidiabetic agents. The compounds of this invention may be combinedwith one or more of these compounds to treat diabetes.

Potassium channel antagonists are also utilized as Class 3anti-arrhythmic agents and to treat acute infarctions in humans. Anumber of naturally occuring toxins are known to block potassiumchannels including Apamin, Iberiotoxin, Charybdotoxin, Noxiustoxin,Kaliotoxin, Dendrotoxin(s), mast cell degranuating (MCD) peptide, andβ-Bungarotoxin (β-BTX). The compounds of this invention may be combinedwith one or more of these compounds to treat arrhythmias.

Depression is related to a decrease in neurotransmitter release. Currenttreatments of depression include blockers of neurotransmitter uptake,and inhibitors of enzymes involved in neurotransmitter degradation whichact to prolong the lifetime of neurotransmitters.

Alzheimer's disease is also characterized by a diminishedneuro-transmitter release. Alzheimer's disease is a neurodegenerativedisease of the brain leading to severely impaired cognition andfunctionality. This disease leads to progressive regression of memoryand learned functions. Alzheimer's disease is a complex disease thataffects cholinergic neurons, as well as serotonergic, noradrenergic andother central neurotransmitter systems. Manifestations of Alzheimer'sdisease extend beyond memory loss and include personality changes,neuromuscular changes, seizures, and occasionally psychotic features.

Alzheimer's disease is the most common type of dementia in the UnitedStates. Some estimates suggest that up to 47% of those older than 85years have Alzheimer's disease. Since the average age of the populationis on the increase, the frequency of Alzheimer's disease is increasingand requires urgent attention. Alzheimer's is a difficult medicalproblem because there are presently no adequate methods available forits prevention or treatment.

Three classes of drugs are being investigated for the treatment ofAlzheimer's disease. The first class consists of compounds that augmentacetyl-choline neurotransmitter function. Currently, cholinergicpotentiators such as the anticholinesterase drugs are being used in thetreatment of Alzheimer's disease. In particular, physostigmine(eserine), an inhibitor of acetylcholinesterase, has been used in itstreatment. The administration of physostigmine has the drawback of beingconsiderably limited by its short half-life of effect, poor oralbioavailability, and severe dose-limiting side-effects, particularlytowards the digestive system. Tacrine (tetrahydroaminocridine) isanother cholinesterase inhibitor that has been employed; however, thiscompound may cause hepatotoxicity.

A second class of drugs that are being investigated for the treatment ofAlzheimer's disease is nootropics that affect neuron metabolism withlittle effect elsewhere. These drugs improve nerve cell function byincreasing neuron metabolic activity. Piracetam is a nootropic that maybe useful in combination with acetyl-choline precursors and may benefitAlzheimer's patients who retain some quantity of functionalacetylcholine release in neurons. Oxiracetam is another related drugthat has been investigated for Alzheimer treatment.

A third class of drugs is those drugs that affect brain vasculature. Amixture of ergoloid mesylates is used for the treatment of dementia.Ergoloid mesylates decrease vascular resistance and thereby increasecerebral blood flow. Also employed are calcium channel blocking drugsincluding Nimodipine which is a selective calcium channel blocker thataffects primarily brain vasculature.

Other miscellaneous drugs are targeted to modify other defects found inAlzheimer's disease. Selegiline, a monoamine oxidase B inhibitor whichincreases brain dopamine and norepinephrine has reportedly caused mildimprovement in some Alzheimer's patients. Aluminum chelating agents havebeen of interest to those who believe Alzheimer's disease is due toaluminum toxicity. Drugs that affect behavior, includeing neuroleptics,and anxiolytics have been employed. Side effects of neuroleptics rangefrom drowsiness and anti cholinergic effects to extrapyramidal sideeffects; other side effects of these drugs include seizures,inappropriate secretion of antidiuretic hormone, jaundice, weight gainand increased confusion. Anxiolytics, which are mild tranquilizers, areless effective than neuroleptics, but also have milder side effects. Useof these behavior-affecting drugs, however, remains controversial. Thepresent invention is related to novel compounds which are useful aspotassium channel antagonists. It is believed that certain diseases suchas depression, memory disorders and Alzheimers disease are the result ofan impairment in neurotransmitter release. The potassium channelantagonists of the present invention may therefore be utilized as cellexcitants which should stimulate an unspecific release ofneurotransmitters such as acetylcholine, serotonin and dopamine.Enhanced neurotransmitter release should reverse the symptoms associatedwith depression and Alzheimers disease.

The compounds within the scope of the present invention exhibitpotassium channel antagonist activity and thus are useful in disordersassociated with potassium channel malfunction. A number of cognitivedisorders such as Alzheimer's Disease, memory loss or depression maybenefit from enhanced release of neuro-transmitters such as serotonin,dopamine or acetylcholine and the like. Blockage of Maxi-K channelsmaintains cellular depolarization and therefore enhances secretion ofthese vital neurotransmitters.

The compounds of this invention may be combined with anticholin-esterasedrugs such as physostigmine (eserine) and Tacrine(tetrahydroaminocridine), nootropics such as Piracetam, Oxiracetam,ergoloid mesylates, selective calcium channel blockers such asNimodipine, or monoamine oxidase B inhibitors such as Selegiline, in thetreatment of Alzheimer's disease. The compounds of this invention mayalso be combined with Apamin, Iberiotoxin, Charybdotoxin, Noxiustoxin,Kaliotoxin, Dendrotoxin(s), mast cell degranuating (MCD) peptide,β-Bungarotoxin (β-BTX) or a combination thereof in treating arrythmias.The compounds of this invention may further be combined with Glyburide,Glipizide, Tolbutamide or a combination thereof to treat diabetes.

The herein examples illustrate but do not limit the claimed invention.Each of the claimed compounds are potassium channel antagonists and arethus useful in the described neurological disorders in which it isdesirable to maintain the cell in a depolarized state to achieve maximalneurotransmitter release. The compounds produced in the presentinvention are readily combined with suitable and known pharmaceuticallyacceptable excipients to produce compositions which may be administeredto mammals, including humans, to achieve effective potassium channelblockage.

The maxi-K channel blockers used in the instant invention can beadministered in a therapeutically effective amount intravaneously,subcutaneously, topically, transdermally, parenterally or any othermethod known to those skilled in the art. Ophthalmic pharmaceuticalcompositions are preferably adapted for topical administration to theeye in the form of solutions, suspensions, ointments, creams or as asolid insert. Ophthalmic formulations of this compound may contain from0.01 to 5% and especially 0.5 to 2% of medicament. Higher dosages as,for example, about 10% or lower dosages can be employed provided thedose is effective in reducing intraocular pressure, treating glaucoma,increasing blood flow velocity or oxygen tension. For a single dose,from between 0.001 to 5.0 mg, preferably 0.005 to 2.0 mg, and especially0.005 to 1.0 mg of the compound can be applied to the human eye.

The pharmaceutical preparation which contains the compound may beconveniently admixed with a non-toxic pharmaceutical organic carrier, orwith a non-toxic pharmaceutical inorganic carrier. Typical ofpharmaceutically acceptable carriers are, for example, water, mixturesof water and water-miscible solvents such as lower alkanols oraralkanols, vegetable oils, peanut oil, polyalkylene glycols, petroleumbased jelly, ethyl cellulose, ethyl oleate, carboxymethyl-cellulose,polyvinylpyrrolidone, isopropyl myristate and other conventionallyemployed acceptable carriers. The pharmaceutical preparation may alsocontain non-toxic auxiliary substances such as emulsifying, preserving,wetting agents, bodying agents and the like, as for example,polyethylene glycols 200, 300, 400 and 600, carbowaxes 1,000, 1,500,4,000, 6,000 and 10,000, antibacterial components such as quaternaryammonium compounds, phenylmercuric salts known to have cold sterilizingproperties and which are non-injurious in use, thimerosal, methyl andpropyl paraben, benzyl alcohol, phenyl ethanol, buffering ingredientssuch as sodium borate, sodium acetates, gluconate buffers, and otherconventional ingredients such as sorbitan monolaurate, triethanolamine,oleate, polyoxyethylene sorbitan monopalmitylate, dioctyl sodiumsulfosuccinate, monothioglycerol, thiosorbitol, ethylenediaminetetracetic acid, and the like. Additionally, suitable ophthalmicvehicles can be used as carrier media for the present purpose includingconventional phosphate buffer vehicle systems, isotonic boric acidvehicles, isotonic sodium chloride vehicles, isotonic sodium boratevehicles and the like. The pharmaceutical preparation may also be in theform of a microparticle formulation. The pharmaceutical preparation mayalso be in the form of a solid insert. For example, one may use a solidwater soluble polymer as the carrier for the medicament. The polymerused to form the insert may be any water soluble non-toxic polymer, forexample, cellulose derivatives such as methylcellulose, sodiumcarboxymethyl cellulose, (hydroxyloweralkyl cellulose), hydroxyethylcellulose, hydroxypropyl cellulose, hydroxypropylmethyl cellulose;acrylates such as polyacrylic acid salts, ethylacrylates,polyactylamides; natural products such as gelatin, alginates, pectins,tragacanth, karaya, chondrus, agar, acacia; the starch derivatives suchas starch acetate, hydroxymethyl starch ethers, hydroxypropyl starch, aswell as other synthetic derivatives such as polyvinyl alcohol, polyvinylpyrrolidone, polyvinyl methyl ether, polyethylene oxide, neutralizedcarbopol and xanthan gum, gellan gum, and mixtures of said polymer.

Suitable subjects for the administration of the formulation of thepresent invention include primates, man and other animals, particularlyman and domesticated animals such as cats and dogs.

The pharmaceutical preparation may contain non-toxic auxiliarysubstances such as antibacterial components which are non-injurious inuse, for example, thimerosal, benzalkonium chloride, methyl and propylparaben, benzyldodecinium bromide, benzyl alcohol, or phenylethanol;buffering ingredients such as sodium chloride, sodium borate, sodiumacetate, sodium citrate, or gluconate buffers; and other conventionalingredients such as sorbitan monolaurate, triethanolamine,polyoxyethylene sorbitan monopalmitylate, ethylenediamine tetraaceticacid, and the like.

The ophthalmic solution or suspension may be administered as often asnecessary to maintain an acceptable IOP level in the eye. It iscontemplated that administration to the mammalian eye will be about onceor twice daily.

For topical ocular administration the novel formulations of thisinvention may take the form of solutions, gels, ointments, suspensionsor solid inserts, formulated so that a unit dosage comprises atherapeutically effective amount of the active component or somemultiple thereof in the case of a combination therapy.

Methodologies for making the compounds of this invention can be gleanedfrom WO 9512603, WO9635712, EP 322633, JO 3002-117 and U.S. Pat. No.3,246,025. In particular, the compounds of this invention can be made,with some modification, in accordance with U.S. Pat. No. 5,629,343 andWO 9616027, which are incorporated herein by reference. The followingexamples, given by way of illustration, is demonstrative of the presentinvention.

EXAMPLE 1 Diethyl2-[2-(phenyl)ethyl]malonate

According to the described procedure (J. Med. Chem., 1984, 27, 967-978),to a 2 liter 3-neck round bottom flask was attached a pressure equalizedaddition funnel and a water cooled reflux condenser. A large Tefloncoated magnetic stirring bar was added and the flask flamed dried undernitrogen. Sodium hydroxide (60% in mineral oil, 42 g, 1.06 moles) wasadded to the flask and the mineral oil removed by washing the solidthree times with 40 mL of hexane. One liter of dry tetrahydrofuran (THF)(distilled over potassium/benzophenone) was added to the flask undernitrogen. A mineral oil bubbler was attached by a rubber septum to theflask and diethyl malonate was added dropwise over 1 h, keeping thetemperature <18° C. When hydrogen evolution had ceased, 107.5 grams of1-bromoethyl benzene (1.06 mole) was added dropwise over 30 minutes withcooling (<30°). The solution was gently refluxed overnight. The mixturewas cooled to room temperature and poured into 200 mL of ice watercontaining 150 mmole of HCl and stirred 5 minutes. The organic layer wasseparated and the aqueous layer extracted with 3×100 mL of ether. Theorganic layers were combined and washed with 2×100 mL of saturatedsodium bicarbonate and 1×100 mL of brine. The solution was dried overanhydrous MgSO4. The ether was removed under reduced pressure and theproduct distilled under vacuum (bp=146-148 @ 1.5 mm Hg). Recovered 203 gof product (yield=72%).

Monoethyl 2-[2-(phenyl)ethyl]malonate acid

Following the reported procedure (J. Med. Chem, 1982, 24, 109-113),diethyl 2-[2-(phenyl)ethyl-malonate (200 g, 0.756 mole) was dissolved in600 mL of absolute ethanol in a 2 liter round bottom flask fitted with aTeflon coated stirring bar. The solution was cooled to 5° C., in an icebath. Then 49.9 g of potassium hydroxide (85%) in 600 mL of absoluteethanol was added dropwise to the rapidly stirring solution over a 2hour period with the solution temperature kept under 15° C. The solutionwas stirred under nitrogen overnight at 25° C. Ethanol was removed underreduced pressure and the syrupy residue dissolved in 800 mL of icewater. The solution was washed with 2×200 ml of ether, the aqueous layeracidified with concentrated HCl (pH<3) and extracted 3×300 mL of ether.The combined ether layer was washed with brine and dried over anhydrousMgSO4. The solution was filtered and the ether removed under reducedpressure to give a clear oil. Recovered 162 g of product (90% yield).

2-Methylene-4-phenylbutyric acid, ethyl ester

Monoethyl 2-[2-(phenyl)ethyl]malonic acid (47.2 g, 200 mole) was addedto 40 mL round bottom flask fitted with a Teflon coated stirring bar.Piperidine (3 mL) and paraformaldehyde (8.4 g, 280 mmole) were added tothe flask and the flask was heated to 55-60° C., until gas evolutionceased. TLC indicated that no starting material remained. The solutionwas worked up by removing the solvent under reduced pressure. Water (50mL) and enough 12N HCl was added to the flask to acidify the mixture(pH>3). The mixture was extracted with ether (3×50 mL) and backextracted with brine. The solution was dried over MgSO₄ and filtered.The reaction mixture was shown to be clean by NMR and TLC and usedwithout further purification. Recovered 36.3 g material (89% yield).

2-Methylene-4-phenylbutyric acid

4-phenyl-2-methylenebutyric acid, ethyl ester (10 g, 49 mmole) wasdissolved in 100 mL of absolute ethanol, Potassium hydroxide (3.32 g,85%, 50 mmole) was added to the solution and the solution stirredovernight at room temperature. After 18 hours, all of the ester washydolyzed. The solvent was removed under reduced pressure and the syrupdissolved in water. Enough concentrated HCl was added to acidify thesolution (pH>3) and the product was extracted with ether (3×50 mL). Thesolution was dried over MgSO₄, filtered and the solvent removed underreduced pressure. Recovered 8.6 g of product (97% yield).

2-[Acetylthiomethyl]-4-phenylbutyric acid

A 250 mL round bottomed flask, fitted with a Teflon magnetic stirringbar and a reflux condenser, was filled with 150 mL of toluene, 0.5 mLpiperidine and 8.6 g (49 mmole) of 4-phenyl-2-methylenebutyric acid.Then 4.1 g (54 mmole) of thiolacetic acid was added to the solution andthe mixture heated to reflux for 8 hours under nitrogen. ¹H-NMR analysisof the reaction mixture indicated some starting material remained.Another 1 g of thiolacetic acid was added and the mixture stirred atreflux for an additional 4 hours. By ¹H-NMR, all of the startingmaterial was seen to have reacted. The solution was cooled to 5° C., and100 mL of ether added to the mixture. The excess thiolacetic acid wasremoved by extraction (3×50 mL 2% sodium bicarbonate). Then 50 mL of 1NHCl was added to the organic layer and the solution shaken vigorously.The organic layer was separated and dried over MgSO₄. The solution wasfiltered and the solvent removed under reduced pressure. Recovered 10.0g of product (81% yield).

N-(2-Acetylthiomethyl-4-phenylbutanoyl)-(L)-Leucine t-butyl ester

To a solution of 4-phenyl-2-[acetylthio-methyl]butyric acid (3.57 g,14.2 mmole) in 66 mL of THF at 0° C., was added (S)-leucine, t-butylester (2.91 g 15.6 mmole) and 1-hydroxybenzotriazole hydrate (HOBTH₂O,2.86 g, 21.2 mmole) and N-methylmorpholine (4.29 g, 42.4 mmole). Themixture was stirred at 0° C. for 15 minutes, then 5.42 g of1-ethyl-3-(3-dimethyl-aminopropyl) carbodiimide hydrochloride (EDC′HCl,28.3 mmol) was added and the mixture stirred overnight. The solution wasworked up by adding 100 mL methylene chloride and the mixture extractedwith 3×50 mL of 5% sodium bicarbonate and washed with 2×50 mL of brine.The organic layer was dried over MgSO4, filtered, and the solventremoved under reduced pressure. The residue was purified by flashchromatography on silica gel eluted with methylene chloride/ethylacetate (95/5). The product was separated as diastereomeric (R,S) and(S,S) fractions. The absolute stereochemistry of each fraction was notdetermined. Higher Rf material=1.78 g; lower Rf material=1.90 g.

N-(2-Acetylthiomethyl-4-phenylbutanoyl)-(L)-leucine, N-phenylamide

To a solution of N-(2-acetylthiomethyl-4-phenylbutanoyl)-(L)-leucine(derived from the higher Rf TLC fraction) (182 mg, 0.5 mmole) in 2 mL ofTHF at 0° C. was added aniline (93 mg, 1.0 mmole) and1-hydroxybenzotriazole hydrate (HOBT′H₂O), 101 mg, 0.75 mole) andN-methylmorpholine (202 mg, 2.0 mmole). The mixture was stirred at 0° C.for 15 minutes, then 192 mg of1-ethyl-3-(3-dimethyl-aminopropyl)carbodiimide hydrochloride (EDC.HCl,1.0 mmole) was added and the mixture stirred overnight. The solution wasworked up by adding 7 mL methylene chloride and the mixture extractedwith 3×3 ml of 5% sodium bicarbonate, then 2×2 mL of brine. The organiclayer was dried over MgSO4, filtered and the solvent removed underreduced pressure. The residue was purified by flash columnchromatography on silica gel eluted with methylene chloride/ethylacetate (90/10) to obtain 110 mg of product (yield=50%).

Functional Assays

A. Maxi-K Channel—TsA-201 Cells

The identification of inhibitors of the Maxi-K channel can beaccomplished using Aurora Biosciences technology, and is based on theability of expressed Maxi-K channels to set cellular resting potentialafter transient transfection of both α and β1 subunits of the channel inTsA-201 cells. In the absence of inhibitors, cells display ahyperpolarized membrane potential, negative inside, close to E_(K) (−80mV) which is a consequence of the activity of the Maxi-K channel.Blockade of the Maxi-K channel will cause cell depolarization. Changesin membrane potential can be determined with voltage-sensitivefluorescence resonance energy transfer (FRET) dye pairs that use twocomponents, a donor coumarin (CC₂DMPE) and an acceptor oxanol(DiSBAC₂(3)). Oxanol is a lipophilic anion and distributes across themembrane according to membrane potential. Under normal conditions, whenthe inside of the cell is negative with respect to the outside, oxanolis accumulated at the outer leaflet of the membrane and excitation ofcoumarin will cause FRET to occur. Conditions that lead to membranedepolarization will cause the oxanol to redistribute to the inside ofthe cell, and, as a consequence, to a decrease in FRET. Thus, the ratiochange (donor/acceptor) increases after membrane depolarization.

Transient transfection of the Maxi-K channel in TsA-201 cells can becarried out as previously described (Hanner et al. (1998) J. Biol. Chem.273, 16289-16296) using FUGENE6™ as the transfection reagent. Twentyfour hours after transfection, cells are collected in Ca²⁺—Mg²⁺-freeDulbecco's phosphate-buffered saline (D-PBS), subjected tocentrifugation, plated onto 96-well poly-d-lysine coated plates at adensity of 60,000 cells/well, and incubated overnight. The cells arethen washed 1× with D-PBS, and loaded with 100 μl of 4 μM CC₂DMPE-0.02%pluronic-127 in D-PBS. Cells are incubated at room temperature for 30min in the dark. Afterwards, cells are washed 2× with D-PBS and loadedwith 100 μl of 6 μM DiSBAC₂(3) in (mM): 140 NaCl, 0.1 KCl, 2 CaCl₂, 1MgCl₂, 20 Hepes-NaOH, pH 7.4, 10 glucose. Test compounds are dilutedinto this solution, and added at the same time. Cells are incubated atroom temperature for 30 min in the dark.

Plates are loaded into a voltage/ion probe reader (VIPR) instrument, andthe fluorescence emission of both CC₂DMPE and DiSBAC₂(3) are recordedfor 10 sec. At this point, 100 μl of high-potassium solution (mM): 140KCl, 2 CaCl₂, 1 MgCl₂, 20 Hepes-KOH, pH 7.4, 10 glucose are added andthe fluorescence emission of both dyes recorded for an additional 10sec. The ratio CC₂DMPE/DiSBAC₂(3), before addition of high-potassiumsolution equals 1. In the absence of any inhibitor, the ratio afteraddition of high-potassium solution varies between 1.65-2.2. When theMaxi-K channel has been completely inhibited by either a known standardor test compound, this ratio remains at 1. It is possible, therefore, totitrate the activity of a Maxi-K channel inhibitor by monitoring theconcentration-dependent change in the fluorescence ratio.

The compounds of this invention were found to causeconcentration-dependent inhibition of the fluorescence ratio with IC₅₀'sin the range of about 10 nM to about 5 μM, more preferably from about100 nM to about 1 μM.

B. Maxi-K Channel Assay—HEK-293 Cells

Under appropriate conditions the maxi-K channel sets the restingpotential of the HEK-293 cells stably transfected with this channel.Generally, addition of high-potassium solution causes the cells todepolarize and this activity can be monitored with fluorescence dyesusing a voltage/ion probe reader (VIPR) instrument. Preincubation of thecells with an inhibitor of the maxi-K channel will lead to celldepolarization. Under these conditions, addition of the high-potassiumsolution will not cause any change in the emission properties of thefluorescence dyes because the cells are already predepolarized. BecauseHEK-293 cells have endogenous potassium conductances, these conductanceshave to be eliminated so that the maxi-K channel is the predominate onesetting the resting potential at E_(k). Elimination is achieved when theHEK-293 cells are incubated with a potassium channel blocker prior toadding a test compound. The consequence of this pharmacologicalmanipulation is the generation of a very large screening window wherethe fluorescence signal denoting a hyperpolarized resting potential isabolished by selective maxi-K channel blockers.

Preferred potassium channel blockers are those that selectivelyeliminate the endogenous potassium conductances of the HEK-293 cellswithout affecting maxi-K channel activity. Untransfected HEK-293 cellsare commercially available. The HEK-293 cells can be transfected asdescribed herein.

The identification of inhibitors of the Maxi-K channel is based on theability of expressed Maxi-K channels to set cellular resting potentialafter transfection of both alpha and beta 1 subunits of the channel inHEK-293 cells and after being incubated with potassium channel blockersthat selectively eliminate the endogenous potassium conductances ofHEK-293 cells. In the absence of maxi-K channel inhibitors, thetransfected HEK-293 cells display a hyperpolarized membrane potential,negative inside, close to E_(K) (−80 mV) which is a consequence of theactivity of the maxi-K channel. Blockade of the Maxi-K channel byincubation with maxi-K channel blockers will cause cell depolarization.Changes in membrane potential can be determined with voltage-sensitivefluorescence resonance energy transfer (FRET) dye pairs that use twocomponents, a donor coumarin (CC₂DMPE) and an acceptor oxanol(DiSBAC₂(3)).

Oxanol is a lipophilic anion and distributes across the membraneaccording to membrane potential. Under normal conditions, when theinside of the cell is negative with respect to the outside, oxanol isaccumulated at the outer leaflet of the membrane and excitation ofcoumarin will cause FRET to occur. Conditions that lead to membranedepolarization will cause the oxanol to redistribute to the inside ofthe cell, and, as a consequence, to a decrease in FRET. Thus, the ratiochange (donor/acceptor) increases after membrane depolarization, whichdetermines if a test compound actively blocks the maxi-K channel.

The HEK-293 cells were obtained from the American Type CultureCollection, 12301 Parklawn Drive, Rockville, Md., 20852 under accessionnumber ATCC CRL-1573. Any restrictions relating to public access to thecell lines shall be irrevocably removed upon patent issuance.

Transfection of the alpha and betal subunits of the maxi-K channel inHEK-293 cells was carried out as follows: HEK-293 cells were plated in100 mm tissue culture treated dishes at a density of 3×10⁶ cells perdish, and a total of five dishes were prepared. Cells were grown in amedium consisting of Dulbecco's Modified Eagle Medium (DMEM)supplemented with 10% Fetal Bovine serum, 1× L-Glutamine, and 1×Penicillin/Streptomycin, at 37° C., 10% CO₂. For transfection withMaxi-K hα (pCIneo) and Maxi-K hβ1 (pIRESpuro) DNAs, 150 μl FuGENE6™ wasadded dropwise into 10 ml of serum free/phenol-red free DMEM and allowedto incubate at room temperature for 5 minutes. Then, the FuGENE6™solution was added dropwise to a DNA solution containing 25 μg of eachplasmid DNA, and incubated at room temperature for 30 minutes. After theincubation period, 2 ml of the FuGENE6™/DNA solution was added dropwiseto each plate of cells and the cells were allowed to grow two days underthe same conditions as described above. At the end of the second day,cells were put under selection media which consisted of DMEMsupplemented with both 600 μg/ml G418 and 0.75 μg/ml puromycin. Cellswere grown until separate colonies were formed. Five colonies werecollected and transferred to a 6 well tissue culture treated dish. Atotal of 75 colonies were collected. Cells were allowed to grow until aconfluent monolayer was obtained. Cells were then tested for thepresence of maxi-K channel alpha and betal subunits using an assay thatmonitors binding of ¹²⁵I-iberiotoxin-D19Y/Y36F to the channel. Cellsexpressing ¹²⁵I-iberiotoxin-D19Y/Y36F binding activity were thenevaluated in a functional assay that monitors the capability of maxi-Kchannels to control the membrane potential of transfected HEK-293 cellsusing fluorescence resonance energy transfer (FRET) ABS technology witha VIPR instrument. The colony giving the largest signal to noise ratiowas subjected to limiting dilution. For this, cells were resuspended atapproximately 5 cells/ml, and 200 μl were plated in individual wells ina 96 well tissue culture treated plate, to add ca. one cell per well. Atotal of two 96 well plates were made. When a confluent monolayer wasformed, the cells were transferred to 6 well tissue culture treatedplates. A total of 62 wells were transferred. When a confluent monolayerwas obtained, cells were tested using the FRET-functional assay.Transfected cells giving the best signal to noise ratio were identifiedand used in subsequent functional assays.

-   -   1. To measure binding of ¹²⁵I-iberiotoxin-D19Y/Y36F to        transfected HEK-293 cells, cells were plated in poly-D-lysine        treated 96 wells at a density of 40,000 cells/well. Cells were        grown overnight under selection medium. Then, the medium is        removed and 200 μl of a solution containing about 70 pM        ¹²⁵I-iberiotoxin-D19Y/Y36F in selection medium is added per        well. For determination of nonspecific binding the same medium        also contained 100 nM unlabeled iberiotoxin. Cells are incubated        with this solution for four hours at 37° C., 10% CO₂. After        incubation, radioactive medium is removed and cells are washed        one time with D-PBS. Then, 200 μl of Microscint-20 is added to        each well and radioactivity associated with the cells is        determined in a Packard Topcount instrument.

The transfected cells (2E+06 Cells/mL) are then plated on 96-wellpoly-D-lysine plates at a density of about 100,000 cells/well andincubated for about 16 to about 24 hours. The medium is aspirated of thecells and the cells washed one time with 100 μl of Dulbecco's phosphatebuffered saline (D-PBS). One hundred microliters of about 9 μM coumarin(CC₂DMPE)-0.02% pluronic-127 in D-PBS per well is added and the wellsare incubated in the dark for about 30 minutes. The cells are washed twotimes with 100 μl of Dulbecco's phosphate-buffered saline and 100 μl ofabout 4.5 μM of oxanol (DiSBAC₂(3)) in (mM) 140 NaCl, 0.1 KCl, 2 CaCl₂,1 MgCl₂, 20 Hepes-NaOH, pH 7.4, 10 glucose is added. Three micromolar ofan inhibitor of endogenous potassium conductance of HEK-293 cells suchas Compounds A or B (see below) is added. A maxi-K channel blocker isadded (about 3 micromolar to about 10 micromolar) and the cells areincubated at room temperature in the dark for about 30 minutes.

The plates are loaded into a voltage/ion probe reader (VIPR) instrument,and the fluorescence emission of both CC₂DMPE and DiSBAC₂(3) arerecorded for 10 sec. At this point, 100 μl of high-potassium solution(mM): 140 KCl, 2 CaCl₂, 1 MgCl₂, 20 Hepes-KOH, pH 7.4, 10 glucose areadded and the fluorescence emission of both dyes recorded for anadditional 10 sec. The ratio CC₂DMPE/DiSBAC₂(3), before addition ofhigh-potassium solution equals 1. In the absence of maxi-K channelinhibitor, the ratio after addition of high-potassium solution variesbetween 1.65-2.2. When the Maxi-K channel has been completely inhibitedby either a known standard such as compounds 1-4 or test compound, thisratio remains at 1. It is possible, therefore, to titrate the activityof a Maxi-K channel inhibitor by monitoring the concentration-dependentchange in the fluorescence ratio.Compounds A and B are:

C. Electrophysiological Assays of Compound Effects on High-conductanceCalcium-activated Potassium Channels

Human Non-pigmented Ciliary Epithelial Cells

The activity of high-conductance calcium-activated potassium (maxi-K)channels in human non-pigmented ciliary epithelial cells was determinedusing electrophysiological methods. Currents through maxi-K channelswere recorded in the inside-out configuration of the patch clamptechnique, where the pipette solution faces the extracellular side ofthe channel and the bath solution faces the intracellular side. Excisedpatches contained one to about fifty maxi-K channels. Maxi-K channelswere identified by their large single channel conductance (250-300 pS),and by sensitivity of channel gating to membrane potential andintracellular calcium concentration. Membrane currents were recordedusing standard electrophysiological techniques. Glass pipettes (Garner7052) were pulled in two stages with a Kopf puller (model 750), andelectrode resistance was 1-3 megohms when filled with saline. Membranecurrents were recorded with EPC9 (HEKA Instruments) or Axopatch ID (AxonInstruments) amplifiers, and digital conversion was done with ITC-16interfaces (Instrutech Corp). Pipettes were filled with (mM); 150 KCl,10 Hepes, 1 MgCl₂, 0.01 CaCl₂, 3.65 KOH, pH 7.20. The bath(intracellular) solution was identical, except, in some cases, calciumwas removed, 1 mM EGTA was added and 20 mM KCl was replaced with 20 mMKF to eliminate calcium to test for calcium sensitivity of channelgating. Drugs were applied to the intracellular side of the channel bybath perfusion.

Human non-pigmented ciliary epithelial cells were grown in tissueculture as described (Martin-Vasallo, P., Ghosh, S., and Coca-Prados,M., 1989, J. Cell. Physiol. 141, 243-252), and plated onto glass coverslips prior to use. High resistance seals (>1 Gohm) were formed betweenthe pipette and cell surface, and inside out patches were excised.Maxi-K channels in the patch were identified by their gating properties;channel open probability increased in response to membranedepolarization and elevated intracellular calcium. In patches used forpharmacological analysis, removing intracellular calcium eliminatedvoltage-gated currents. Maxi-K currents were measured after depolarizingvoltage steps or ramps that caused channel opening.

The compounds of this invention were applied to the intracellular sideof the channel in appropriate concentrations (0.001 to 10 μM). Thecompounds reduced channel open probability, and this effect was reversedupon washout of compounds from the experimental chamber. The IC50 forblock of maxi-K channels under these conditions for the compounds ofthis invention ranged from about 0.5 nM to about 300 nM.

1. A compound selected from the group consisting of:


2. A method for treating ocular hypertension or glaucoma comprisingadministration to a patient in need of such treatment a therapeuticallyeffective amount of a compound of structural formula I:

or a pharmaceutically acceptable salt, enantiomer, diastereomer ormixture thereof: wherein, R and R² independently represent C₁₋₆ alkyl,(CH₂)_(n)aryl, (CH₂)_(n)heteroaryl, (CH₂)_(n)heterocycloalkyl, saidalkyl, aryl or heteroaryl optionally substituted with 1-3 groups of R³;Y represents —(CH₂)_(n)SCOR⁴; X represents CH₂, or O (in which m doesnot exist); R³ represents hydrogen, C₁₋₆ alkoxy, C₁₋₆ alkyl, CF₃, nitro,amino, cyano, C₁₋₆ alkylamino, or halogen and R⁴ represents C₁₋₆ alkoxy,or C₁₋₆ alkyl; R⁷ represents H, halo, OH, NO₂, NH₂, CN, alkoxy, —COO—,alkoxycarbonyl, haloalkyl, alkoxycarbonylalkyl, or alkylsulphonyl mrepresents 1-3; n represents 0-3.
 3. The method according to claim 2wherein the compound of formula I is applied as a topical formulation.4. A method according to claim 2 wherein R is C₁₋₆ alkyl, or (CH₂)_(n)aryl, and all other variables are as originally described.
 5. A methodaccording to claim 2 wherein R₂ is C₁₋₆ alkyl, or (CH₂)_(n)aryl, and allother variables are as originally described.
 6. A method according toclaim 2 wherein X is CH₂ and all other variables are as originallydescribed.
 7. A method according to claim 2 wherein Y is —(CH₂)_(n)SCOR⁴wherein n=0, and all other variables are as originally described.
 8. Amethod according to claim 2 wherein Y is —(CH₂)_(n)SCOR⁴, n=1-3, and allother variables are as originally described.
 9. A method according toclaim 2 wherein R is (CH₂)_(n) aryl, R² is C₁₋₆ alkyl, Y is(CH₂)_(n)SCOR⁴, X is CH₂ and m=1.
 10. A method according to claim 2wherein R is (CH₂)_(n)aryl, R² is C₁₋₆ alkyl, Y is (CH₂)_(n)SCOR⁴, X isCH₂ and m=2.
 11. A method according to claim 2 wherein R is C₁₋₆ alkyl,R² is (CH₂)_(n)aryl, Y is (CH₂)_(n) SCOR⁴, X is CH₂ and m=2.
 12. Amethod according to claim 2 wherein R is (CH₂)_(n)aryl, R² is(CH₂)_(n)aryl, Y is (CH₂)_(n) SCOR⁴, X is CH₂ and m=2.
 13. A methodaccording to claim 3 wherein the topical formulation is a solution orsuspension.
 14. A method according to claim 3 wherein an activeingredient belonging to the group consisting of: β-adrenergic blockingagent, parasympathomimetic agent, carbonic anhydrase inhibitor, and aprostaglandin or a prostaglandin derivative is optionally added to theformulation.
 15. A method according to claim 14 wherein the β-adrenergicblocking agent is timolol; the parasympathomimetic agent is pilocarpine;the carbonic anhydrase inhibitor is dorzolamide, acetazolamide,metazolamide or brinzolamide; the prostaglandin is latanoprost orrescula, and the prostaglandin derivative is a hypotensive lipid derivedfrom PGF2α prostaglandins.
 16. A method for treating macular edema ormacular degeneration comprising administration to a patient in need ofsuch treatment a pharmaceutically effective amount of a compound ofstructural formula I:

or a pharmaceutically acceptable salt, enantiomer, diastereomer ormixture thereof: wherein, R and R² independently represent C₁₋₆ alkyl,(CH₂)_(n)aryl, (CH₂)_(n)heteroaryl, (CH₂)_(n)heterocycloalkyl, saidalkyl, aryl or heteroaryl optionally substituted with 1-3 groups of R³;Y represents —(CH₂)_(n)SCOR⁴; X represents CH₂, or O (in which m doesnot exist); R³ represents hydrogen, C₁₋₆ alkoxy, C₁₋₆ alkyl, CF₃, nitro,amino, cyano, C₁₋₆ alkylamino, or halogen and R⁴ represents C₁₋₆ alkoxy,or C₁₋₆ alkyl; R⁷ represents H, halo, OH, NO₂, NH₂, CN, alkoxy, —COO—,alkoxycarbonyl, haloalkyl, alkoxycarbonylalkyl, or alkylsulphonyl; mrepresents 1-3; and/or n represents 0-3.
 17. The method according toclaim 16 wherein the compound of formula I is applied as a topicalformulation.
 18. A method according to claim 16 wherein the compound is:


19. A method for increasing retinal and optic nerve head blood velocityor increasing retinal and optic nerve oxygen tension comprisingadministration to a patient in need of such treatment an effectiveocular hypertensive formulation containing a potassium channel blockerof structural formula I:

or a pharmaceutically acceptable salt, enantiomer, diastereomer ormixture thereof: wherein, R and R² independently represent C₁₋₆ alkyl,(CH₂)_(n)aryl, (CH₂)_(n)heteroaryl, (CH₂)_(n)heterocycloalkyl, saidalkyl, aryl or heteroaryl optionally substituted with 1-3 groups of R³;Y represents —(CH₂)_(n)SCOR⁴; X represents CH₂, or O (in which m doesnot exist); R³ represents hydrogen, C₁₋₆ alkoxy, C₁₋₆ alkyl, CF₃, nitro,amino, cyano, C₁₋₆ alkylamino, or halogen and R⁴ represents C₁₋₆ alkoxy,or C₁₋₆ alkyl; R⁷ represents H, halo, OH, NO₂, NH₂, CN, alkoxy, —COO—,alkoxycarbonyl, haloalkyl, alkoxycarbonylalkyl, or alkylsulphonyl; mrepresents 1-3; and/or n represents 0-3.
 20. The method according toclaim 19 wherein the compound of formula I is applied as a topicalformulation.
 21. A method according to claim 19 wherein the compound is:


22. A method for providing a neuroprotective effect comprisingadministration to a patient in need of such treatment a therapeuticallyeffective amount of structural formula I:

or a pharmaceutically acceptable salt, enantiomer, diastereomer ormixture thereof: wherein, R and R² independently represent C₁₋₆ alkyl,(CH₂)_(n)aryl, (CH₂)_(n)heteroaryl, (CH₂)_(n)heterocycloalkyl, saidalkyl, aryl or heteroaryl optionally substituted with 1-3 groups of R³;Y represents —(CH₂)_(n)SCOR⁴; X represents CH₂, or O (in which m doesnot exist); R³ represents hydrogen, C₁₋₆ alkoxy, C₁₋₆ alkyl, CF₃, nitro,amino, cyano, C₁₋₆ alkylamino, or halogen and R⁴ represents C₁₋₆ alkoxy,or C₁₋₆ alkyl; R⁷ represents H, halo, OH, NO₂, NH₂, CN, alkoxy, —COO—,alkoxycarbonyl, haloalkyl, alkoxycarbonylalkyl, or alkylsulphonyl; mrepresents 1-3; and/or n represents 0-3.
 23. The method according toclaim 22 wherein the compound of formula I is applied as a topicalformulation.
 24. A method according to claim 22 wherein the compound is:


25. A method according to claim 2 in which the topical formulationoptionally contains xanthan gum or gellan gum.
 26. A method for treatingocular hypertension or glaucoma comprising administration to a patientin need of such treatment a therapeutically effective amount ofstructural formula I:

or a pharmaceutically acceptable salt, enantiomer, diastereomer ormixture thereof: wherein, R and R² independently represent C₁₋₆ alkyl,(CH₂)_(n)aryl, (CH₂)_(n)heteroaryl, (CH₂)_(n)heterocycloalkyl, saidalkyl, aryl or heteroaryl optionally substituted with 1-3 groups of R³;Y represents —(CH₂)_(n)SCOR⁴; X represents CH₂, or O (in which m doesnot exist); R³ represents hydrogen, C₁₋₆ alkoxy, C₁₋₆ alkyl, CF₃, nitro,amino, cyano, C₁₋₆ alkylamino, or halogen and R⁴ represents C₁₋₆ alkoxy,or C₁₋₆ alkyl; R⁷ represents H, halo, OH, NO₂, NH₂, CN, alkoxy, —COO—,alkoxycarbonyl, haloalkyl, alkoxycarbonylalkyl, or alkylsulphonyl; mrepresents 1-3; and/or n represents 0-3.
 27. A method according to claim26 wherein the compound is:


28. A method of preventing repolarization or hyperpolarization of amammalian cell wherein the cell contains a potassium channel comprisingthe administration to a mammal, including a human, in need thereof, of apharmacologically effective amount of a potassium channel blocker ofstructural formula I:

or a pharmaceutically acceptable salt, enantiomer, diastereomer ormixture thereof: wherein, R and R² independently represent C₁₋₆ alkyl,(CH₂)_(n)aryl, (CH₂)_(n)heteroaryl, (CH₂)_(n)heterocycloalkyl, saidalkyl, aryl or heteroaryl optionally substituted with 1-3 groups of R³;Y represents —(CH₂)_(n)SCOR⁴; X represents CH₂, or O (in which m doesnot exist); R³ represents hydrogen, C₁₋₆ alkoxy, C₁₋₆ alkyl, CF₃, nitro,amino, cyano, C₁₋₆ alkylamino, or halogen and R⁴ represents C₁₋₆ alkoxy,or C₁₋₆ alkyl; R⁷ represents H, halo, OH, NO₂, NH₂, CN, alkoxy, —COO—,alkoxycarbonyl, haloalkyl, alkoxycarbonylalkyl, or alkylsulphonyl; mrepresents 1-3; and/or(?) n represents 0-3.
 29. A method of treatingdepression in a patient in need thereof comprising administering apharmaceutically effective amount of a compound according to claim 28.30. A method of treating cognitive disorders in a patient in needthereof comprising administering a pharmaceutically effective amount ofa compound according to claim
 28. 31. A method of treating arrhythmiadisorders in a patient in need thereof comprising administering apharmaceutically effective amount of a compound according to claim 28.32. A method of treating diabetes in a patient in need thereofcomprising administering a pharmaceutically effective amount of acompound according to claim 28.